SUMMARY Melanocytic nevi are benign growths of melanocytes that initiate spontaneously, reach a fixed size, and eventually regress. While most nevi are benign, they can be difficult to distinguish from melanoma, leading to the unnecessary excision of many normal nevi. Conventional wisdom suggests that nevi arrest their growth by a mechanism called ?oncogene-induced senescence?- nevi initiate when melanocytes acquire a BRAF mutation, this clonal population of melanocytes replicate for a fixed number of cell cycles, and eventually nevi just stop growing. All cells in nevi, however, cannot be truly senescent; nevi can regrow after partial excision, can be induced to grow with UV irradiation, and some nevi can even evolve into melanoma. Little is known about how this growth arrest is achieved in vivo. We recently used a combination of multiphoton microscopy (MPM) imaging and single cell sequencing to better understand the evolution of BRAF mutant melanocytic nevi in mouse skin. These studies revealed that nevi are composed of two populations of melanocytes: a population with less pigment that expresses stem cell markers (nevus-initiating cells-NI), and a well- differentiated population of melanocytes full of melanin (nevus arrested cells-NA). Single cell gene expression analysis revealed that NI cells expressed Wnt receptors/ Wnt downstream targets while NA cells expressed secreted Wnt inhibitors, identifying a feedback loop that could restrict nevus growth. The stem cell like population also expressed cytokines that recruit macrophages, and large numbers of infiltrating macrophages were observed in nevus skin. These observations led us to hypothesize that nevi develop from a NI cells that: 1) generates pigmented progeny that then restricts their growth; 2) recruits macrophages that ultimately facilitate nevus regression. In this proposal, we use a combination of single cell genomics, live imaging, genetically engineered mouse models, and archived human tissues to: 1) determine whether nevi initiate from a nevus initiating cell that expresses stem cell markers; 2) test whether nevi arrest their growth secondary to a Wnt inhibitor feedback loop that restricts the growth of the nevus initiating cell population; 3) test whether nevi regress secondary to infiltrating macrophages. Completion of these studies will not only lead to a better understanding of the origin and evolution of melanocytic nevi, but also define a set of molecular markers that can be used to identify those nevi with malignant potential that need to be removed.